(20R)-2a-Methyl-19,26,27-Trinor-Vitamin D Analogs

ABSTRACT

This invention discloses (20R)-2α-methyl-19,26,27-trinor-vitamin D analogs, and specifically (20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D 3 , and pharmaceutical uses therefor. This compound exhibits transcription activity as well as pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. This compound also shows very low activity in vivo on bone calcium mobilization and intestinal calcium transport activity compared to the native hormone 1α,25-dihydroxyvitamin D 3 , and therefore may be used to treat autoimmune disorders or inflammatory diseases in humans as well as renal osteodystrophy. This compound may also be used for the treatment or prevention of obesity.

CROSS REFERENCE TO RELATED APPLICATIONS

The present patent application is a continuation-in-part of U.S.application Ser. No. 11/697,434 filed Apr. 6, 2007, which applicationclaims priority to U.S. Provisional Patent Application No. 60/744,386filed Apr. 6, 2006.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to(20R)-2α-methyl-19,26,27-trinor-vitamin D analogs and theirpharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)),as have analogs with a cyclopropyl group in the side chain (e.g. MC-903known as calcipotriene and described in Nishii et al U.S. Pat. No.5,063,221).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1-hydroxy-2-methylene-19-nor-homopregnacalciferol isdescribed in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

17-ene vitamin D compounds as well as vitamin D compounds having adouble bond in the side chain thereof are also known, and have beenproposed for various pharmacological uses. Bone diseases such asosteoporosis, skin disorders such as psoriasis, cancers such as leukemiaand cosmetic conditions such as wrinkles are just some of theapplications proposed for such compounds. 17-ene compounds are describedin U.S. Pat. Nos. 5,545,633; 5,929,056 and 6,399,797 while 2-alkylidenecompounds having a side chain with a double bond therein are describedin, for example, U.S. Pat. No. 5,843,928.

19-nor vitamin D compounds substituted at the carbon-2 position of ringA with an alkyl group such as methyl, or an alkylidene group such asmethylene, and having a side chain lacking one or more of the standardvitamin D₃ substitutents, are also known, and have been proposed forvarious pharmacological uses. For example, numerous2α-methyl-19,26,27-trinor analogs are described in published U.S.Application No. 2007/028704 and in published U.S. Application No.2007/0270391, and numerous 2-methylene-19,26,27-trinor analogs aredescribed in published U.S. Application No. 2007/0249567. In addition,2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol is describedin published U.S. Application No. 2007/0254857.

SUMMARY OF THE INVENTION

The present invention is directed toward(20R)-2α-methyl-19,26,27-trinor-vitamin D analogs, their biologicalactivity, and various pharmaceutical uses for these compounds. These newvitamin D compounds not known heretofore are the 19-nor-vitamin Danalogs having a methyl group at the 2-position (C-2), a hydroxylsubstituent attached to the 25-position (C-25) in the side chain, andthe methyl groups normally located at the 26 and 27 positions (C-26 andC-27) in the side chain replaced with hydrogen atoms. The preferredvitamin D analog is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ (hereinafterreferred to as “B-9”).

Structurally these (20R)-2α-methyl-19,26,27-trinor-vitamin D analogs arecharacterized by the general formula I shown below:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group. The preferredanalog is (20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃which has the following formula Ia:

The above compounds I, particularly Ia, exhibit a desired, and highlyadvantageous, pattern of biological activity. These compounds arecharacterized by relatively high binding to vitamin D receptors, whichis only slightly less than that of the native hormone1α,25-dihydroxyvitamin D₃. These compounds have very little, if any,ability to promote intestinal calcium transport in vivo. They would beclassified as having substantially no potency in vivo in stimulatingintestinal calcium transport activity, as compared to that of1α,25-dihydroxyvitamin D₃. These compounds I, and particularly Ia, alsohave very little ability to mobilize calcium from bone, and they wouldbe classified as having substantially no bone calcium mobilizingactivity as compared to 1α,25-dihydroxyvitamin D₃. It is undesirable toraise serum calcium to supraphysiologic levels when suppressing thepreproparathyroid hormone gene (Darwish & DeLuca, Arch. Biochem.Biophys. 365, 123-130, 1999) and parathyroid gland proliferation. Theseanalogs having relatively no bone calcium mobilization activity whilevery active on cell differentiation are expected to be useful as atherapy for suppression of secondary hyperparathyroidism of renalosteodystrophy.

The compounds I, particularly Ia, of the invention have also beendiscovered to be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compounds of the invention.

The above compounds I, and particularly Ia, are also characterized byrelatively high cell differentiation activity and in promotingtranscription. Thus, these compounds also provide a therapeutic agentfor the treatment of psoriasis, or as an anti-cancer agent, especiallyagainst leukemia, colon cancer, breast cancer, skin cancer and prostatecancer. In addition, due to their relatively high cell differentiationactivity, these compounds provide a therapeutic agent for the treatmentof various skin conditions including wrinkles, lack of adequate dermalhydration, i.e. dry skin, lack of adequate skin firmness, i.e. slackskin, and insufficient sebum secretion. Use of these compounds thus notonly results in moisturizing of skin but also improves the barrierfunction of skin.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of one or more of the compounds or thepharmaceutical compositions to the subject inhibits adipocytedifferentiation, inhibits gene transcription, and/or reduces body fat inthe animal subject.

One or more of the compounds may be present in a composition to treatthe above-noted diseases and disorders in an amount from about 0.01μg/gm to about 1000 μg/gm of the composition, preferably from about 0.1μg/gm to about 500 μg/gm of the composition, and may be administeredtopically, transdermally, orally, rectally, nasally, sublingually orparenterally in dosages of from about 0.01 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIGS. 1-5 illustrate various biological activities of(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃, hereinafterreferred to as “B-9”, as compared to the native hormone1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of B-9 and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of B-9 and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to B-9;

FIG. 4 is a graph illustrating the bone calcium mobilization activity of1,25(OH)₂D₃ as compared to B-9 in a group of animals; and

FIG. 5 is a graph illustrating the intestinal calcium transport activityof 1,25(OH)₂D₃ as compared to B-9 in a group of animals.

DETAILED DESCRIPTION OF THE INVENTION

(20R)-1α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ (referred toherein as “B-9”) a 19-nor vitamin D analog which is characterized by thepresence of a methyl substituent at the carbon 2 (C-2), a hydroxylsubstituent attached to the 25-position (C-25) in the side chain, andthe methyl groups normally located at the 26 and 27 positions (C-26 andC-27) in the side chain replaced with hydrogen atoms, was synthesizedand tested. Such vitamin D analog seemed an interesting target becausethe relatively small methyl group at the C-2 position should notinterfere with binding to the vitamin D receptor. Structurally, this19-nor analog is characterized by the general formula Ia previouslyillustrated herein, and its pro-drug (in protected hydroxy form) ischaracterized by general formula I previously illustrated herein.

The preparation of (20R)-2α-methyl-19,26,27-trinor-vitamin D analogshaving the structure I can be accomplished by a common general method,i.e. the condensation of a bicyclic Windaus-Grundmann type ketone IIwith the allylic phosphine oxide III to the corresponding2-methylene-19-nor-vitamin D analog IV followed by deprotection at C-1,C-3 and C-25 in the latter compound (see Scheme III herein):

Thereafter, the 2-methylene substituent is converted to a mixture of the2α-methyl and 2β-methyl epimers, and finally the 2α-methyl analog Ia maybe separated and obtained from the mixture by reversed-phase HPLC. Inthe structures II, III and IV, groups X₁, X₂ and X₃ arehydroxy-protecting groups, preferably t-butyldimethylsilyl, it beingalso understood that any functionalities that might be sensitive, orthat interfere with the condensation reaction, be suitably protected asis well-known in the art. The process shown above represents anapplication of the convergent synthesis concept, which has been appliedeffectively for the preparation of vitamin D compounds [e.g. Lythgoe etal., J. Chem. Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev.9, 449 (1983); Toh et al., J. Org. Chem. 48, 1414 (1983); Baggiolini etal., J. Org. Chem. 51, 3098 (1986); Sardina et al., J. Org. Chem. 51,1264 (1986); J. Org. Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No.5,086,191; DeLuca et al., U.S. Pat. No. 5,536,713].

The hydrindanone of the general structure II is not known. It can beprepared by the method shown in Schemes I and II herein (see thepreparation of compound B-9).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compounds I and Ia isillustrated and described more completely in U.S. Pat. No. 5,945,410entitled “2-Alkyl-19-Nor-Vitamin D Compounds” the specification of whichis specifically incorporated herein by reference.

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

More specifically, reference should be made to the followingillustrative example and description as well as to Schemes I, II and IIIherein for a detailed illustration of the preparation of compound B-9.

In this example specific products identified by Arabic numerals (1, 2,3) refer to the specific structures so identified in the Schemes I, II,and III.

EXAMPLE

Chemistry. Ultraviolet (UV) absorption spectra were recorded with aHitachi Model 60-100 UV-vis spectrometer in the solvent noted. ¹Hnuclear magnetic resonance (NMR) spectra were recorded at 500 MHz with aBruker AM-500 FT spectrometer in deuteriochloroform. Chemical shifts (δ)are reported downfield from internal Me₄Si (δ 0.00). Mass spectra wererecorded at 70 eV on a Kratos DS-50 TC instrument equipped with a KratosMS-55 data system. Samples were introduced into the ion sourcemaintained at 120-250° C. via a direct insertion probe. High-performanceliquid chromatography (HPLC) was performed on a Waters Associates liquidchromatograph equipped with a Model 6000A solvent delivery system, aModel 6 UK Universal injector, a Model 486 tunable absorbance detector,and a differential R 401 refractometer.

Example 1

Preparation of (8S,20S)-de-A,B-20-(hydroxymethyl)-pregnan-8-ol (1).Ozone was passed through a solution of vitamin D₂ (3 g, 7.6 mmol) inmethanol (250 mL) and pyridine (2.44 g, 2.5 mL, 31 mmol) for 50 min at−78° C. The reaction mixture was then flushed with an oxygen for 15 minto remove the residual ozone and the solution was treated with NaBH₄(0.75 g, 20 mmol). After 20 min the second portion of NaBH₄ (0.75 g, 20mmol) was added and the mixture was allowed to warm to room temperature.The third portion of NaBH₄ (0.75 g, 20 mmol) was then added and thereaction mixture was stirred for 18 h. The reaction was quenched withwater (40 mL) and the solution was concentrated under reduced pressure.The residue was extracted with ethyl acetate (3×80 mL) and the combinedorganic phase was washed with 1M aq. HCl, saturated aq. NaHCO₃, dried(Na₂SO₄) and concentrated under reduced pressure. The residue waschromatographed on silica gel with hexane/ethyl acetate (75:25) to givethe diol 1 (1.21 g, 75% yield) as white crystals.

m.p. 106-108° C.; [α]_(D)+30.2° (c 1.46, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ 4.08 (1H, d, J=2.0 Hz, 8α-H), 3.63 (1H, dd, J=10.5, 3.1 Hz, 22-H),3.38 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.99 (1H, br.d, J=13.2 Hz), 1.03(3H, d, J=6.6 Hz, 21-H₃), 0.956 (3H, s, 18-H₃); ¹³C NMR (100 MHz) δ69.16 (d, C-8), 67.74 (t, C-22), 52.90 (d), 52.33 (d), 41.83 (s, C-13),40.19 (t), 38.20 (d), 33.53 (t), 26.62 (t), 22.54 (t), 17.36 (t), 16.59(q, C-21), 13.54 (q, C-18); MS (EI) m/z 212 (2, M⁺), 194 (34, M⁺-H₂O),179 (33, M⁺-H₂O—CH₃), 163 (18, M⁺-CH₂OH—H₂O), 135 (36), 125 (54), 111(100), 95 (63), 81 (67); exact mass calculated for C₁₃H₂₂O (M⁺-H₂O)194.1671, found 194.1665.

Preparation of (8S,20S)-de-A,B-20-(acetyloxymethyl)-pregnan-8-ol (2).Acetic anhydride (1.05 mL, 1.13 g, 11.1 mmol) was added to a solution ofthe diol 1 (1.8 g, 8.5 mmol) and triethylamine (4.2 mL, 3.03 g, 30 mmol)in anhydrous dichloromethane (10 mL) at 0° C. The mixture was stirredunder argon at room temperature for 18 h. The reaction was quenched withwater (10 mL) and extracted with dichloromethane. The combined organicphase was washed with brine, dried (Na₂SO₄) and concentrated underreduced pressure. The pure alcohol 2 (2.09 g, 97% yield) was isolated bya chromatography on silica gel with hexane/ethyl acetate (95:5, then9:1), as a colorless oil:

[α]_(D)+34.4° (c 1.63, CHCl₃); ¹H NMR (500 MHz, CDCl₃+TMS) δ 4.09 (1H,s, 8α-H), 4.07 (1H, dd, J=10.7, 3.5 Hz, 22-H), 3.78 (1H, dd, J=10.7, 7.5Hz, 22-H), 2.05 (3H, s, COMe), 1.99 (1H, dm, J=12.8 Hz), 1.00 (3H, d,J=6.6 Hz, 21-H₃), 0.96 (3H, s, 18-H₃); ¹³C NMR (125 MHz) δ 171.35 (s,C═O), 69.39 (t, C-22), 69.07 (d, C-8), 53.21 (d), 52.30 (d), 41.91 (s,C-13), 40.19 (t), 35.30 (d), 33.54 (t), 26.60 (t), 22.53 (t), 20.95 (q,COMe), 17.36 (t), 16.97 (q, C-21), 13.50 (q, C-18); MS (EI) m/z 254 (10,M⁺), 236 (24, M⁺-H₂O), 212 (9, M⁺-C₂H₂O), 194 (67, M⁺-CH₃COOH), 176 (91,M⁺-CH₃COOH—H₂O), 161 (83), 150 (80), 135 (91), 125 (97), 112 (98), 97(100); exact mass calculated for C₁₅H₂₄O₂ (M⁺-H₂O) 235.1776, found236.1771.

Preparation of(8S,20S)-de-A,B-8-triethylsilyloxy-20-(acetyloxymethyl)-pregnane (3).Triethylsilyl trifluoromethanesulfonate (1.9 mL, 2.24 g, 8.5 mmol) wasadded to a solution of the alcohol 2 (2.08 g, 8.2 mmol) and 2,6-lutidine(2.86 mL, 2.63 g, 24.6 mmol) in anhydrous dichloromethane (10 mL) at 0°C. The mixture was stirred under argon at 0° C. for 0.5 h. The reactionwas quenched with water (30 mL) and extracted with dichloromethane. Thecombined organic phase was washed with water, dried (Na₂SO₄) andconcentrated under reduced pressure. The product 3 (2.9 g, 96% yield)was isolated by a chromatography on silica gel with hexane/ethyl acetate(95:5), as a colorless oil:

[α]_(D)+40.8° (c 1.95, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.06 (1H, dd,J=10.7, 3.4 Hz, 22-H), 4.04 (1H, s, 8α-H), 3.77 (1H, dd, J=10.7, 7.6 Hz,22-H, 2.04 (3H, s, COMe), 1.93 (1H, dm, J=12.4 Hz), 0.98 (3H, d, J=6.6Hz, 21-H₃), 0.94 (9H, t, J=7.9 Hz, Si(CH₂CH ₃)₃), 0.92 (3H, s, 18-H₃),0.55 (6H, q, J=7.9 Hz, Si(CH₂ CH₃)₃); ¹³C NMR (100 MHz) δ 171.39 (s,C═O), 69.57 (t, C-22), 69.22 (d, C-8), 53.41 (d), 52.81 (d), 42.22 (s,C-13), 40.60 (t), 35.36 (d), 34.59 (t), 26.77 (t), 23.04 (t), 20.99 (q,COMe), 17.64 (t), 17.05 (q, C-21), 13.53 (q, C-18), 6.92 (q, SiCH₂ CH₃),4.93 (t, SiCH₂CH₃); MS (EI) m/z 368 (29, M⁺), 339 (85, M⁺-C₂H₅), 325(78, M⁺-CH₃CO), 265 (31, M⁺-CH₃COOC₃H₇—H), 237 (21, M⁺-Et₃SiO), 217(88), 189 (72), 177 (92), 161 (77), 145 (93), 135 (98), 121 (90), 107(91), 95 (100); exact mass calculated for C₁₉H₃₅O₃Si (M⁺-C₂H₅) 339.2355,found 339.2352.

Preparation of(8S,20S)-de-A,B-8-triethylsilyloxy-20-(hydroxymethyl)-pregnane (4). Asolution of sodium hydroxide (1.5 g, 37.5 mmol) in anhydrous ethanol (20mL) was added to a mixture of the compound 3 (2.9 g, 7.9 mmol) inanhydrous ethanol (10 mL). The reaction mixture was stirred at roomtemperature for 30 min. and then neutralized with 5% aq. hydrochloricacid. The mixture was extracted with dichloromethane and the combinedorganic phases were washed with water, dried (Na₂SO₄) and concentratedunder reduced pressure. The residue was chromatographed on silica gelwith hexane/ethyl acetate (95:5, then 9:1) to give the alcohol 4 (2.58g, 100% yield):

[α]_(D)+38.9° (c 2.45, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.07 (1H, d,J=2.3 Hz, 8α-H), 3.66 (1H, dd, J=10.5, 3.2, Hz, 22-H), 3.39 (1H, dd,J=10.5, 6.8 Hz, 22-H), 1.98 (1H, dm, J=12.7 Hz), 1.05 (3H, d, J=6.6 Hz,21-H₃),0.98 (9H, t, J=7.9 Hz, Si(CH₂CH ₃)₃), 0.95 (3H, s, 18-H₃), 0.58(6H, q, J=7.9 Hz, Si(CH₂ CH₃)₃); ¹³C NMR (125 MHz) δ 69.26 (d, C-8),67.97 (t, C-22), 53.10 (d), 52.86 (d), 42.14 (s, C-13), 40.63 (t), 38.28(d), 34.60 (t), 26.80 (t), 23.05 (t), 17.64 (t), 16.65 (q, C-21), 13.56(q, C-18), 6.91 (q, SiCH₂ CH₃), 4.92 (t, SiCH₂CH₃); MS (EI) m/z 326 (58,M⁺), 311 (15, M⁺-CH₃), 297 (93, M⁺-C₂H₅), 283 (89, M⁺-C₂H₂O), 225 (80),211 (24, M⁺-Et₃Si), 193 (90), 177 (98), 135 (98), 121 (99), 107 (99), 95(100); exact mass calculated for C₁₉H₃₈O₂Si (M⁺) 326.2641, found326.2549.

Preparation of(8S,20S)-de-A,B-8-triethylsilyloxy-20-(iodomethyl)-pregnane (5). Asolution of iodine (1.52 g, 6 mmol) in methylene chloride (120 mL) wasslowly added to a solution of triphenylphosphine (1.6 g, 6.1 mmol) andimidazole (816 mg, 12 mmol) in methylene chloride (10 mL) at 0 ° C.After 15 min. a solution of alcohol 4 (0.5 g, 1.5 mmol) in methylenechloride (10 mL) was added, the mixture was stirred at 0° C. for 20 min.and at room temperature for 18 h. The reaction mixture was washed withwater, dried (Na₂SO₄) and concentrated under reduced pressure. Theresidue was chromatographed on silica gel with hexane to give thedesired iodide 5 (657 mg, 100%):

[α]_(D)+52.0° (c 1.44, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.04 (1H, d,J=2.1 Hz, 8α-H), 3.33 (1H, dd, J=9.5, 2.3 Hz, 22-H), 3.17 (1H, dd,J=9.5, 5.3 Hz), 1.90 (1H, dm, J=12.5 Hz), 0.99 (3H, d, J=5.9 Hz, 21-H₃),0.95 (9H, t, J=7.9 Hz, Si(CH₂CH ₃)₃), 0.95 (3H, s, 18-H₃), 0.55 (6H, q,J=7.9 Hz, Si(CH ₂CH₃)₃); ¹³C NMR (100 MHz) δ 69.25 (d, C-8), 56.03 (d),52.80 (d), 42.10 (s, C-13), 40.46 (t), 36.46 (d), 34.50 (t), 26.66 (t),22.85 (t), 21.61 (t), 20.71 (q, C-21), 17.61 (t), 14.34 (q, C-18), 6.94(q, SiCH₂ CH₃), 4.93 (t, SiCH₂CH₃); MS (EI) m/z 436 (42, M⁺), 421 (3,M⁺-CH₃), 407 (95, M⁺-C₂H₉), 393 (76), 309 (23, M⁺-I), 303 (86,M⁺-Et₃SiOH—H), 251 (28), 225 (35), 177 (96), 135 (96), 121 (87), 95(97), 75 (100); exact mass calculated for C₁₉H₃₇OSiI (M⁺) 436.1658,found 436.1645.

Preparation of(8S,20R)-de-A,B-8-triethylsilyloxy-20-(3-isopropoxycarbonyl)-propyl-pregnane(6). A mixture of zinc powder (488 mg, 7.5 mmol), anhydrous pyridine (8mL) and isopropyl acrylate (900 μL, 855 mg, 7.5 mmol) was warmed to 50°C., then nickel(II) chloride hexahydrate (428 mg, 1.8 mmol) was added.The resulting mixture was warmed to 65° C. and stirred for 2 h until itsgreen color turned to reddish brown one. After cooling to 0° C., asolution of iodide 5 (657 mg, 1.5 mmol) in anhydrous pyridine (6 mL) wasadded and the reaction mixture was stirred for 7 h at room temperature.The mixture was diluted with ethyl acetate (20 mL) and the resultingprecipitate was filtered off through a pad of Celite. The filtrate waswashed with 5% aq. HCl and brine, dried (Na₂SO₄) and concentrated underreduced pressure. The residue was chromatographed on silica gel withhexane and hexane/ethyl acetate (95:5) to give the ester 6 (494 mg,78%): [α]_(D)+41.8° (c 1.41, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.99 (1H,sep, J=6.3 Hz, OCHMe₂), 4.01 (1H, s, 8α-H), 2.22 (2H, m, 24-H₂), 1.93(1H, dm, J=12.2 Hz), 1.22 (6H, d, J=6.3 Hz, OCHMe₂ ), 0.93 (9H, t, J=7.9Hz, Si(CH₂CH ₃)₃), 0.89 (3H, d, J=5.6 Hz, 21-H₃), 0.88 (3H, s, 18-H₃),0.54 (6H, q, J=7.9 Hz, Si(CH ₂CH₃)₃); ¹³C NMR (125 MHz) δ 173.44 (s,COO-iPr), 69.37 (d, C-8), 67.26 (d, COOCHMe₂), 56.50 (d), 53.06 (d),42.09 (s, C-13), 40.74 (t), 35.20 (t), 35.13 (t), 35.01 (d), 34.63 (t),27.25 (t), 22.98 (t), 21.84 (q, COOCHMe₂ ), 21.58 (t), 18.52 (q, C-21),17.66 (t), 13.46 (q, C-18), 6.92 (q, SiCH₂ CH₃), 4.91 (t, SiCH₂CH₃); MS(EI) m/z 424 (35, M⁺), 409 (6, M⁺-CH₃), 395 (87, M⁺-C₂H₉), 381 (39,M⁺-C₃H₇), 365 (41, M⁺-C₃H₇O), 335 (90, M⁺-C₃H₇COOH—H), 295 (29), 249(26), 225 (64), 215 (54), 199 (33), 171 (29), 135 (88), 115 (55), 103(100); exact mass calculated for C₂₅H₄₈O₃Si (M⁺) 424.3373, found424.3373.

Preparation of(8S,20R)-de-A,B-8-triethylsilyloxy-20-(4-hydroxy-butyl)-pregnane (7).Lithium aluminium hydride (19 mg, 0.5 mmol) was added to a solution ofester 6 (100 mg, 0.24 mmol) in anhydrous THF (8 mL) at 0° C. A coolingbath was removed and the reaction mixture was stirred for 18 h at roomtemperature. The excess hydride was quenched by careful, successiveaddition of methanol. A saturated aq. solution of the tartaric acid wasadded and the mixture was extracted with methylene chloride. Thecombined organic phase was washed with water, dried (Na₂SO₄) andconcentrated under reduced pressure. The residue was chromatographed onsilica gel with hexane/ethyl acetate (95:5) to give the alcohol 7 (87mg, 99%):

[α]_(D)+46.7° (c 0.78, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.02 (1H, d,J=2.0 Hz, 8α-H), 3.63 (2H, t, J=6.6 Hz, 25-H₂), 1.95 (1H, dm, J=12.6Hz), 0.95 (9H, t, J=7.9 Hz, Si(CH₂CH ₃)₃), 0.90 (3H, s, 18-H₃), 0.89(3H, d, J=7.9 Hz, 21-H₃), 0.55 (6H, q, J=7.9 Hz, Si(CH₂ CH₃)₃); ¹³C NMR(125 MHz) δ 69.38 (d, C-8), 63.07 (t, C-25), 56.68 (d), 53.08 (d), 42.09(s, C-13), 40.77 (t), 35.54 (t), 35.20 (d), 34.63 (t), 33.26 (t), 27.29(t), 22.98 (t), 22.17 (t), 18.53 (q, C-21), 17.67 (t), 13.47 (q, C-18),6.91 (q, SiCH₂ CH₃), 4.91 (t, SiCH₂CH₃); MS (EI) m/z 368 (8, M⁺), 353(4, M⁺-CH₃), 339 (56, M⁺-C₂H₇), 325 (53), 297 (18), 283 (13), 225 (54),177 (37), 163 (69), 135 (93), 103 (100); exact mass calculated forC₂₀H₃₉O₂Si (M⁺-C₂H₅) 399.2719, found 339.2713.

Preparation of (8S,20R)-de-A,B-20-(4-hydroxy-butyl)-pregnan-8-ol (8). Toa solution of compound 7 (86 mg, 0.23 mmol) in tetrahydrofuran (2 mL)and acetonitrile (2 mL) a mixture of aq. 48% HF/acetonitrile (1:9 ratio,2 mL) was added at 0° C. and the resulting mixture was stirred at roomtemperature for 1 h. Saturated aq. NaHCO₃ solution was added and thereaction mixture was extracted with ethyl acetate. The combined organicphase was washed with brine, dried (Na₂SO₄) and concentrated underreduced pressure. The residue was chromatographed on silica gel withhexane/ethyl acetate (9:1) to give the diol 8 (60 mg, 100%):

[α]_(D)+40.2° (c 3.05, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.06 (1H, d,J=2.3 Hz, 8α-H), 3.62 (2H, t, J=6.6 Hz, 25-H₂), 1.99 (1H, dm, J=13.0Hz), 0.92 (3H, s, 18-H₃), 0.90 (3H, d, J=6.5 Hz, 21-H₃); ¹³C NMR (125MHz) δ 69.28 (d, C-8), 62.89 (t, C-25), 56.47 (d), 52.53 (d), 41.75 (s,C-13), 40.30 (t), 35.45 (t), 35.14 (d), 33.46 (t), 33.16 (t), 27.10 (t),22.44 (t), 22.13 (t), 18.42 (q, C-21), 17.36 (t), 13.43 (q, C-18); MS(EI) m/z 254 (7, M⁺), 236 (8, M⁺-H₂O), 221 (7, M⁺-H₂O—CH₃), 163 (8,M⁺-H₂O—C₄H₈OH), 135 (25, M⁺-H₂O—C₆H₁₂OH), 125 (35), 111 (100), 97 (30);exact mass calculated for C₁₆H₃₀O (M⁺) 254.2246, found 254.2450.

Preparation of(8S,20R)-de-A,B-20-[4-(tert-butyldimethylsilyloxy)-butyl]-pregnan-8-ol(9). tert-Butyldimethylsilyl chloride (47 mg, 0.31 mmol) was added to asolution of the diol 8 (60 mg, 0.23 mmol) and triethylamine (134 μL, 97mg, 0.96 mmol) in anhydrous methylene chloride (4 mL). The mixture wasstirred under argon at room temperature for 18 h. The reaction wasquenched with water and extracted with ethyl acetate. The combinedorganic phase was washed with brine, dried (Na₂SO₄) and concentratedunder reduced pressure. The residue was chromatographed on silica gelwith hexane and hexane/ethyl acetate (98:2) to give the alcohol 9 (84mg, 100%):

[α]_(D)+30.1° (c 3.8, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.05 (1H, d,J=2.2 Hz, 8α-H), 3.58 (2H, t, J=6.6 Hz, 25-H₂), 1.98 (1H, dm, J=12.8Hz), 0.92 (3H, s, 18-H₃), 0.88 (3H, d, 21-H₃) covered by 0.88 (9H, s,Si-t-Bu), 0.04 (6H, s, SiMe₂); ¹³C NMR (125 MHz) δ 69.36 (d, C-8), 63.24(t, C-25), 56.65 (d), 52.60 (d), 41.82 (s, C-13), 40.38 (t), 35.46 (t),35.20 (d), 33.57 (t), 33.29 (t), 27.13 (t), 25.95 (q, SiCMe ₃), 22.49(t), 22.16 (t), 18.44 (q, C-21), 18.33 (s, SiCMe₃), 17.41 (t), 13.49 (q,C-18), −5.28 (q, SiMe₂ ); MS (EI) m/z no M⁺, 311 (1, M⁺-C₄H₉), 293 (3,M⁺-C₄H₉—H₂O), 251 (9, M⁺-t-BuSiMe₂H—H), 219 (32, M⁺-H₂O−t-BuSiMe₂O), 163(54), 149 (30), 135 (55), 123 (63), 109 (100), 95 (76); exact masscalculated for C₁₈H₃₅O₂Si (M⁺-C₄H₉) 311.2406, found 311.2399.

Preparation of(20R)-de-A,B-20-[4-(tert-butyldimethylsilyloxy)-butyl]-pregnan-8-one(10). Pyridinium dichromate (127 mg, 0.34 mmol) was added to a solutionof the alcohol 9 (26 mg, 71 μmol) and pyridinium p-toluenesulfonate (3mg, 12 μmol) in anhydrous methylene chloride (6 mL). The resultingsuspension was stirred at room temperature for 3 h. The reaction mixturewas filtered through a Waters silica Sep-Pak cartridge (5 g) that wasfurther washed with methylene chloride. After removal of solvents theketone 10 (23 mg, 89% yield) was obtained as a colorless oil:[α]_(D)+3.8° (c 1.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 3.59 (2H, t,J=6.4 Hz, 25-H₂), 2.43 (1H, dd, J=11.6, 7.5 Hz), 0.94 (3H, d, J=6.1 Hz,21-H₃), 0.885 (9H, s, Si-t-Bu), 0.626 (3H, s, 18-H₃), 0.039 (6H, s,SiMe₂); ¹³C NMR (100 MHz) δ 212.14 (s, C-8), 63.16 (t, C-25), 61.97 (d),56.65 (d), 49.90 (s, C-13), 40.95 (t), 38.96 (t), 35.43 (d and t), 33.20(t), 27.48 (t), 25.95 (q, SiCMe ₃), 24.05 (t), 22.16 (t), 19.03 (t),18.62 (q, C-21), 18.33 (s, SiCMe₃), 12.45 (q, C-18), −5.27 (q, SiMe₂ );MS (EI) m/z 367 (1, M⁺+H), 351 (2, M⁺-CH₃), 309 (66, M⁺-C₄H₉), 267 (17),217 (39, M⁺-H₂O−t-BuSiMe₂O), 175 (21), 161 (34), 135 (92), 121 (51), 95(49), 75 (100); exact mass calculated for C₁₈H₃₃O₂Si (M⁺-C₄H₉) 309.2250,found 309.2244.

Preparation of(20R)-2-methylene-19,26,27-trinor-1α,25-dihydroxycalciferol (13). To asolution of phosphine oxide 11 (87 mg, 149 μmol) in anhydrous THF (600μL) at −20° C. was slowly added PhLi (1.3 M in cyclohexane-ether, 200μL, 260 μmol) under argon with stirring. The solution turned deeporange. After 30 min the mixture was cooled to −78° C. and a precooled(−78° C.) solution of ketone 10 (18 mg, 49 μmol) in anhydrous THF (200μL) was slowly added. The mixture was stirred under argon at −78° C. for3 h and at 0° C. for 18 h. Ethyl acetate was added, and the organicphase was washed with brine, dried (Na₂SO₄) and evaporated. The residuewas dissolved in hexane and applied on a Waters silica Sep-Pak cartridge(2 g). The cartridge was washed with hexane and hexane/ethyl acetate(99.5:0.5) to give 19-norvitamin derivative 12 (32 mg). The Sep-Pak wasthen washed with hexane/ethyl acetate (96:4) to recover the unchangedC,D-ring ketone 10 (5 mg, 14 μmol), and with ethyl acetate to recoverdiphenylphosphine oxide 11 (55 mg). The protected vitamin 12 was furtherpurified by HPLC (9.4×250 mm Zorbax Sil column, 4 mL/min) usinghexane/2-propanol (99.9:0.1) solvent system. Pure compound 12 (31.55 mg,88% yield) was eluted at R_(t)=4.09 min as a colorless oil:

UV (in hexane) λ_(max) 262.3, 252.0, 243.6 nm; ¹H NMR (500 MHz, CDCl₃) δ6.22 and 5.84 (each 1H, each d, J=11.1 Hz, 6- and 7-H), 4.97 and 4.92(each 1H, each s, ═CH₂), 4.42 (2H, m, 1β- and 3α-H), 3.61 (2H, t, J=6.5Hz, 25-H₂), 2.82 (1H, dm, J=12.1 Hz, 9β-H), 2.52 (1H, dd, J=13.3, 5.9Hz, 10α-H), 2.46 (1H, dd, J=12.6, 4.4 Hz, 4α-H), 2.33 (1H, dm, J=13.3Hz, 10β-H), 2.18 (1H, dd, J=12.6, 8.3 Hz, 41β-H), 1.99 (2H, m), 0.92(3H, d, J=6.4 Hz, 21-H₃), 0.902 (9H, s, Si-t-Bu), 0.899 (9H, s,Si-t-Bu), 0.867 (9H, s, Si-t-Bu), 0.545 (3H, s, 18-H₃), 0.082 (3H, s,SiMe), 0.069 (3H, s, SiMe), 0.056 (6H, s, 2×SiMe), 0.052 (3H, s, SiMe),0.028 (3H, s, SiMe); ¹³C NMR (125 MHz) δ 152.99 (s, C-2), 141.26 (s,C-8), 132.70 (s, C-5), 122.43 (d, C-6), 116.10 (d, C-7), 106.25 (t,═CH₂), 72.54 and 71.63 (each d, C-1 and C-3), 63.31 (t, C-25), 56.58(d), 56.29 (d), 47.62 (t), 45.68 (s, C-13), 40.61 (t), 38.56 (t), 36.09(d), 35.64 (t), 33.32 (t), 28.76 (t), 27.71 (t), 25.99 (q, SiCMe ₃),25.84 (q, SiCMe ₃), 25.78 (q, SiCMe ₃), 23.45 (t), 22.28 (t), 22.22 (t),18.76 (q, C-21), 18.38 (s, SiCMe₃), 18.25 (s, SiCMe₃), 18.16 (s,SiCMe₃), 12.06 (q, C-18), −4.86 and −5.09 and −5.23 (each q, 6×SiMe); MS(EI) m/z no M⁺, 673 (8, M⁺-C₄H₉), 628 (2, M⁺-t-BuMeSiH₂), 598 (100,M⁺-t-BuMe₂SiOH), 556 (9), 541 (4), 496 (3), 366 (42), 257 (10), 234(13), 147 (20); exact mass calculated for C₄₃H₈₂O₃Si₃Na (MNa⁺) 753.5470,found 753.5474.

Protected vitamin 12 (31.54 mg, 43 μmol) was dissolved in THF (3 mL) andacetonitrile (3 mL). A solution of aq. 48% HF in acetonitrile (1:9ratio, 2 mL) was added at 0° C. and the resulting mixture was stirred atroom temperature for 3 h. Saturated aq. NaHCO₃ solution was added andthe reaction mixture was extracted with ethyl acetate. The combinedorganic phase was washed with brine, dried (Na₂SO₄) and concentratedunder reduced pressure. The residue was diluted with 2 mL ofhexane/ethyl acetate (8:2) and applied on a Waters silica Sep-Pakcartridge (2 g). An elution with hexane/ethyl acetate (8:2) and ethylacetate gave the crude product 13 (20 mg). The vitamin 13 was furtherpurified by straight phase HPLC [9.4×250 mm Zorbax Sil column, 5 mL/min,hexane/2-propanol (85:15) solvent system, R_(t)=8.75 min.] and later byreverse phase HPLC [9.4×250 mm Zorbax Eclipse XDB-C18 column, 4 mL/min,methanol/water (85:15) solvent system, R_(t)=7.90 min.] to give acolorless oil (13.15 mg, 79% yield):

UV (in EtOH) λ_(max) 262.2, 252.7, 244.2 nm; ¹H NMR (500 MHz, CDCl₃) δ6.34 and 5.88 (1H and 1H, each d, J=11.1 Hz, 6- and 7-H), 5.10 and 5.08(each 1H, each s, ═CH₂), 4.46 (2H, m, 1β- and 3α-H), 3.64 (2H, dd,J=11.8, 6.1 Hz, 25-H₂), 2.84 (1H, dd, J=13.1, 4.3 Hz, 10β-H), 2.81 (1H,br d, J=15.2 Hz, 9β-H), 2.56 (1H, dd, J=13.3, 3.0 Hz, 4α-H), 2.32 (1H,dd, J=13.3, 6.0 Hz, 4β-H), 2.27 (1H, dd, J=13.1, 8.5 Hz, 10-H), 0.92(3H, d, J=6.3 Hz, 21-H₃), 0.542 (3H, s, 18-H₃); ¹³C NMR (125 MHz) δ151.95 (s, C-2), 143.37 (s, C-8), 130.42 (s, C-5), 124.19 (d, C-6),115.28 (d, C-7), 107.70 (t, ═CH₂), 71.79 and 70.61 (each d, C-1 andC-3), 63.08 (t, C-25), 56.40 (d), 56.30 (d), 45.75 (s, C-13) covered by45.75 (t), 40.42 (t), 38.13 (t), 36.02 (d), 35.63 (t), 33.23 (t), 28.94(t), 27.63 (t), 23.47 (t), 22.25 (t), 22.20 (t), 18.75 (q, C-21), 12.06(q, C-18); MS (EI) m/z 388 (3, M⁺), 334 (1, M⁺−3H₂O), 318 (9,M⁺−3H₂O—CH₄), 272 (9), 252 (27), 250 (28), 239 (8), 196 (99), 194 (100),160 (12); exact mass calculated for C₂₅H₄₀O₃ (M⁺) 388.2977, found388.2961.

Preparation of (20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxycalciferol(14) and (20R)-2β-methyl-19,26,27-trinor-1α,25-dihydroxycalciferol (15).Tris(triphenylphosphine)rhodium (I) chloride (7 mg, 7.6 μmol) was addedto dry benzene (5 mL) presaturated with hydrogen (15 min.). The mixturewas stirred at room temperature until a homogeneous solution was formed(25 min). A solution of vitamin 13 (2.9 mg, 7.5 μmol) in dry benzene (3mL) was then added and the reaction was allowed to proceed under acontinuous stream of hydrogen for 4 h. Benzene was removed under vacuum,the residue was redissolved in hexane/ethyl acetate (1:1) and applied ona Waters silica Sep-Pak cartridge (2 g). A mixture of 2-methyl vitaminswas eluted with the same solvent system. The compounds were furtherpurified by HPLC (9.4×250 mm Zorbax-Sil column, 5 mL/min) usinghexane/2-propanol (85:15) solvent system. The mixture of2-methyl-19-norvitamins 14 and 15 gave a single peak at R_(t)=8.91 min.Separation of both epimers was achieved by reversed-phase HPLC (9.4×250mm Zorbax Eclipse XDB-C18 column, 3 mL/min) using methanol/water (85:15)solvent system. 2β-Methyl vitamin 15 (911 μg, 31% yield) was collectedat R_(t)=8.71 min. and its 2α-epimer 14 (1.055 mg, 36% yield) atR_(t)=9.27 min:

2α-Methyl analog 14: UV (in EtOH) λ_(max) 260.5, 251.5, 243.5 nm; ¹H NMR(500 MHz, CDCl₃) δ 6.37 and 5.82 (1H and 1H, each d, J=11.1 Hz, 6- and7-H), 3.96 (1H, m, 1-H), 3.63 (3H, m, 3α-H and 25-H₂), 2.80 (2H, br m,9β- and 10α-H), 2.60 (1H, dd, J=12.8, 4.4 Hz, 4α-H), 2.22 (1H, br d,J=14.3 Hz, 10β-H), 2.13 (1H, ˜t, J˜11.3 Hz, 4β-H), 1.132 (3H, d, J=6.8Hz, 2α-CH₃), 0.926 (3H, d, J=6.5 Hz, 21-H₃), 0.531 (3H, s, 18-H₃); MS(EI) m/z 390 (100, M⁺), 372 (14, M⁺-H₂O), 357 (4, M⁺-H₂O—CH₃), 339 (3,M⁺−2H₂O—CH₃), 317 (23, M⁺-C₄H₈OH), 289 (76, M⁺-C₆H₁₂OH), 271 (45,M⁺-C₆H₁₂OH—H₂O), 253 (40), 235 (46), 194 (35), 159 (30), 147 (57), 135(70); exact mass calculated for C₂₅H₄₂O₃ (M⁺) 390.3134, found 390.3121.

2β-Methyl analog 15: UV (in EtOH) λ_(max) 260.5, 251.0, 243.5 nm; ¹H NMR(500 MHz, CDCl₃) δ 6.26 and 5.87 (1H and 1H, each d, J=11.2 Hz, 6-H and7-H), 3.90 (1H, m, 3α-H), 3.65 (2H, dd, J=12.2, 6.4 Hz, 25-H₂), 3.50(1H, m, 1β-H), 3.08 (1H, dd, J=12.9, 4.1 Hz, 10β-H), 2.79 (1H, dd,J=12.2, 4.3 Hz, 9β-H), 2.43 (1H, br d, J=ca. 13.7 Hz, 4α-H), 2.34 (1H,dd, J=13.7, 2.6 Hz, 4β-H), 1.142 (3H, d, J=6.8 Hz, 2β-CH₃), 0.930 (3H,d, J=6.4 Hz, 21-H₃), 0.543 (3H, s, 18-H₃); MS (EI) m/z 390 (100, M⁺),372 (17, M⁺-H₂O), 354 (5, M⁺−2H₂O), 339 (3, M⁺−2H₂O—CH₃), 317 (21,M⁺-C₄H₈OH), 289 (62, M⁺-C₆H₁₂OH), 271 (37, M⁺-C₆H₁₂OH—H₂O), 253 (39),247 (33), 235 (46), 194 (32), 159 (29), 147 (53), 135 (71); exact masscalculated for C₂₅H₄₂O₃ (M⁺) 390.3134, found 390.3133.

Biological Activity of (20R)-2α-Methyl-19,26,27-Trinor-Vitamin D Analogs

The introduction of a methyl group to the 2-position, as well as ahydroxyl substituent attached to the 25-position (C-25) in the sidechain, and replacing the methyl groups normally located at the 26 and 27positions (C-26 and C-27) in the side chain with hydrogen atoms bondedto the carbon atom at position 25 in the side chain had a small effecton binding of B-9 to the full length recombinant rat vitamin D receptor,as compared to 1α,25-dihydroxyvitamin D₃. The compound B-9 bound withsomewhat lower affinity to the nuclear vitamin D receptor as compared tothe standard 1,25-(OH)₂D₃ (FIG. 1). It might be expected from theseresults that compound B-9 would have equivalent biological activity.Surprisingly, however, compound B-9 is a highly selective analog withunique biological activity.

FIG. 5 shows that B-9 has very little, if any, ability to increaseintestinal calcium transport activity in vivo, and it clearly hassubstantially lower potency in vivo as compared to that of1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport. B-9 did not stimulateintestinal calcium transport even at doses that were about 50 timeshigher than the dose of 1,25(OH)₂D₃.

FIG. 4 demonstrates that B-9 has very little, if any, bone calciummobilization activity, as compared to 1,25(OH)₂D₃. B-9 demonstrated nobone calcium mobilization activity even at very high doses that wereabout 50 times higher than the dose of 1,25(OH)₂D₃. Thus, B-9 clearlyhas significantly lower potency in mobilizing calcium from bone ascompared to 1,25(OH)₂D₃, at the recommended lower doses.

FIGS. 4 and 5 thus illustrate that B-9 may be characterized as havingsubstantially no intestinal calcium transport activity, andsubstantially no bone calcium mobilization activity.

FIG. 2 illustrates that B-9 is about one log, i.e. ten times less potentthan 1,25(OH)₂D₃ on HL-60 cell differentiation, i.e. causing thedifferentiation of HL-60 cells into monocytes. Although B-9 has lower invitro cell differentiation activity than 1,25(OH)₂D₃, its activity oncell differentiation is still relatively significant. Thus, B-9 may bean excellent candidate for the treatment of psoriasis and cancer,especially against leukemia, colon cancer, breast cancer, skin cancerand prostate cancer. In addition, due to its relatively high celldifferentiation activity, this compound provides a therapeutic agent forthe treatment of various skin conditions including wrinkles, lack ofadequate dermal hydration, i.e. dry skin, lack of adequate skinfirmness, i.e. slack skin, and insufficient sebum secretion. Use of thiscompound thus not only results in moisturizing of skin but also improvesthe barrier function of skin.

FIG. 3 illustrates that in bone cells the compound B-9 is about 30 timesless potent than 1,25(OH)₂D₃ in increasing transcription of the24-hydroxylase gene. Again, as with cell differentiation, although B-9'stranscription activity is less than 1,25(OH)₂D₃, its activity in thisregard is still relatively significant. This result, together with thecell differentiation activity of FIG. 2, suggests that B-9 will be veryeffective in psoriasis because it has direct cellular activity incausing cell differentiation, gene transcription, and in suppressingcell growth. These data also indicate that B-9 may have significantactivity as an anti-cancer agent, especially against leukemia, coloncancer, breast cancer, skin cancer and prostate cancer.

The relatively strong activity of B-9 on HL-60 differentiation suggestsit will be active in suppressing growth of parathyroid glands and in thesuppression of the preproparathyroid gene.

EXPERIMENTAL METHODS

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wereoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.

RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (0.47% Ca)diet+AEK oil for one week followed by Diet 11 (0.02% Ca)+AEK oil for 3weeks. The rats were then switched to a diet containing 0.47% Ca for oneweek followed by two weeks on a diet containing 0.02% Ca. Doseadministration began during the last week on 0.02% calcium diet. Fourconsecutive ip doses were given approximately 24 hours apart.Twenty-four hours after the last dose, blood was collected from thesevered neck and the concentration of serum calcium determined as ameasure of bone calcium mobilization. The first 10 cm of the intestinewas also collected for intestinal calcium transport analysis using theeverted gut sac method.

Interpretation Of Data

Summary of Biological Findings. This compound B-9 binds the VDR withslightly less affinity as the native hormone, and can be considered tobe approximately one log, i.e. about 10 times, less potent than1,25(OH)₂D₃ in this activity. B-9 also displays approximately 10 timesless cell differentiation activity and about 30 times less in vitro genetranscription activity compared to 1,25(OH)₂D₃. However, this compoundshows no activity in the bone and intestine at dose levels about 50times higher or more than the dose levels of 1,25(OH)₂D₃ that showactivity. Because this compound exhibits relatively significant celldifferentiation and transcriptional activity, but no calcemic activity,it might be useful for treating patients with various autoimmunediseases, cancer, renal osteodystrophy, psoriasis or other skindiseases. B-9 might not only be useful in the treatment of the abovelisted diseases, but also in the prevention of the above listeddiseases.

VDR binding, HL60 cell differentiation, and transcription activity. B-9(K_(i)=1×10⁻⁹M) is less active than the natural hormone1α,25-dihydroxyvitamin D₃ (K_(i)=9×10⁻¹¹M) in its ability to competewith [³H]-1,25(OH)₂D₃ for binding to the full-length recombinant ratvitamin D receptor (FIG. 1). B-9 displays about one log, i.e. 10 timesless activity (EC₅₀=3×10⁻⁸M) in its ability (efficacy or potency) topromote HL-60 cell differentiation as compared to 1α,25-dihydroxyvitaminD₃ (EC₅₀=2×10⁻⁹M) (See FIG. 2). Also, compound B-9 (EC₅₀=1×10⁻⁸M) hasabout 30 times less transcriptional activity in bone cells than1α,25-dihydroxyvitamin D₃ (EC₅₀=3×10⁻¹⁰M) (See FIG. 3). These resultssuggest that B-9 will be very effective in psoriasis because it hasdirect cellular activity in causing cell differentiation, genetranscription, and in suppressing cell growth. These data also indicatethat B-9 will have significant activity as an anti-cancer agent,especially against leukemia, colon cancer, breast cancer, skin cancerand prostate cancer, as well as against skin conditions such as dry skin(lack of dermal hydration), undue skin slackness (insufficient skinfirmness), insufficient sebum secretion and wrinkles. It would also beexpected to be very active in suppressing secondary hyperparathyroidism.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of B-9 and 1,25(OH)₂D₃ in intestineand bone were tested. As expected, the native hormone (1,25(OH)₂D₃)increased serum calcium levels at all dosages (FIG. 4). The studyreported in FIG. 4 shows that B-9 has relatively low, or little,activity in mobilizing calcium from bone. Even the administration of35,100 pmol/day of B-9 for 4 consecutive days did not result in anymobilization of bone calcium whereas the native hormone 1,25(OH)₂D₃ hadsignificant activity at 780 pmol/day where a substantial effect wasseen.

Intestinal calcium transport was evaluated in the same group of animalsusing the everted gut sac method (FIG. 5). The study reported in FIG. 5shows that B-9 has little or no intestinal calcium transport activity.Administration of 35,100 pmol/day of B-9 for 4 consecutive days did notresult in any activity.

These results show that the compound B-9 does not promote intestinalcalcium transport even when administered at 35,100 pmol/day. Thus, itmay be concluded that B-9 has little, if any, intestinal calciumtransport activity at the recommended lower doses to that of1,25(OH)₂D₃.

These results illustrate that B-9 is an excellent candidate for numeroushuman therapies as described herein, and that it may be particularlyuseful in a number of circumstances such as suppression of secondaryhyperparathyroidism of renal osteodystrophy, autoimmune diseases,cancer, numerous types of skin conditions, and psoriasis. B-9 is anexcellent candidate for treating psoriasis because: (1) it hassignificant VDR binding, transcription activity and cellulardifferentiation activity; (2) it has little hypercalcemic liability,unlike 1,25(OH)₂D₃; and (3) it is easily synthesized. Since B-9 hassignificant binding activity to the vitamin D receptor, but has littleability to raise blood serum calcium, it may also be particularly usefulfor the treatment of secondary hyperparathyroidism of renalosteodystrophy.

These data also indicate that the compound B-9 of the invention may beespecially suited for treatment and prophylaxis of human disorders whichare characterized by an imbalance in the immune system, e.g. inautoimmune diseases, including multiple sclerosis, lupus, diabetesmellitus, host versus graft rejection, and rejection of organtransplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compound B-9 of the invention.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of the compound or the pharmaceuticalcompositions to the subject inhibits adipocyte differentiation, inhibitsgene transcription, and/or reduces body fat in the animal subject. Theanimal may be a human, a domestic animal such as a dog or a cat, or anagricultural animal, especially those that provide meat for humanconsumption, such as fowl like chickens, turkeys, pheasant or quail, aswell as bovine, ovine, caprine, or porcine animals.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I, particularly B-9, may be formulated forpharmaceutical applications as a solution in innocuous solvents, or asan emulsion, suspension or dispersion in suitable solvents or carriers,or as pills, tablets or capsules, together with solid carriers,according to conventional methods known in the art. Any suchformulations may also contain other pharmaceutically-acceptable andnon-toxic excipients such as stabilizers, anti-oxidants, binders,coloring agents or emulsifying or taste-modifying agents.

The compounds of formula I and particularly B-9, may be administeredorally, topically, parenterally, rectally, nasally, sublingually ortransdermally. The compound is advantageously administered by injectionor by intravenous infusion or suitable sterile solutions, or in the formof liquid or solid doses via the alimentary canal, or in the form ofcreams, ointments, patches, or similar vehicles suitable for transdermalapplications. A dose of from 0.01 μg to 1000 μg per day of the compoundsI, particularly B-9, preferably from about 0.1 μg to about 500 μg perday, is appropriate for prevention and/or treatment purposes, such dosebeing adjusted according to the disease to be treated, its severity andthe response of the subject as is well understood in the art. Since thecompound exhibits specificity of action, each may be suitablyadministered alone, or together with graded doses of another activevitamin D compound—e.g. 1α-hydroxyvitamin D₂ or D₃, or1α,25-dihydroxyvitamin D₃—in situations where different degrees of bonemineral mobilization and calcium transport stimulation is found to beadvantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly B-9, as defined by theabove formula I and Ia as the active ingredient, and a suitable carrier.An effective amount of such compound for use in accordance with thisinvention is from about 0.01 μg to about 1000 μg per gm of composition,preferably from about 0.1 μg to about 500 μg per gram of composition,and may be administered topically, transdermally, orally, rectally,nasally, sublingually, or parenterally in dosages of from about 0.01μg/day to about 1000 μg/day, and preferably from about 0.1 μg/day toabout 500 μg/day.

The compounds I, particularly B-9, may be formulated as creams, lotions,ointments, topical patches, pills, capsules or tablets, suppositories,aerosols, or in liquid form as solutions, emulsions, dispersions, orsuspensions in pharmaceutically innocuous and acceptable solvent oroils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly B-9, may be advantageously administered inamounts sufficient to effect the differentiation of promyelocytes tonormal macrophages. Dosages as described above are suitable, it beingunderstood that the amounts given are to be adjusted in accordance withthe severity of the disease, and the condition and response of thesubject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 2. The compound ofclaim 1 wherein X₃ is hydrogen.
 3. The compound of claim 1 wherein X₁ ishydrogen.
 4. The compound of claim 1 wherein X₁, X₂ and X₃ are allt-butyldimethylsilyl.
 5. A pharmaceutical composition containing aneffective amount of at least one compound as claimed in claim 1 togetherwith a pharmaceutically acceptable excipient.
 6. The pharmaceuticalcomposition of claim 5 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 7. Thepharmaceutical composition of claim 5 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 8.(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


9. A pharmaceutical composition containing an effective amount of(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ together witha pharmaceutically acceptable excipient.
 10. The pharmaceuticalcomposition of claim 9 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 11. Thepharmaceutical composition of claim 9 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 12.A method of treating psoriasis comprising administering to a subjectwith psoriasis an effective amount of a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 13. The method ofclaim 12 wherein the compound is administered orally.
 14. The method ofclaim 12 wherein the compound is administered parenterally.
 15. Themethod of claim 12 wherein the compound is administered transdermally.16. The method of claim 12 wherein the compound is administeredtopically.
 17. The method of claim 12 wherein the compound isadministered rectally.
 18. The method of claim 12 wherein the compoundis administered nasally.
 19. The method of claim 12 wherein the compoundis administered sublingually.
 20. The method of claim 12 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 21. The method of claim 12 wherein the compound is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


22. A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 23. The method ofclaim 22 wherein the compound is administered orally.
 24. The method ofclaim 22 wherein the compound is administered parenterally.
 25. Themethod of claim 22 wherein the compound is administered transdermally.26. The method of claim 22 wherein the compound is administeredrectally.
 27. The method of claim 22 wherein the compound isadministered nasally.
 28. The method of claim 22 wherein the compound isadministered sublingually.
 29. The method of claim 22 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 30. The method of claim 22 wherein the compound is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


31. A method of treating an autoimmune disease selected from the groupconsisting of multiple sclerosis, lupus, diabetes mellitus, host versusgraft rejection, and rejection of organ transplants, comprisingadministering to a subject with said disease an effective amount of acompound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 32. The method ofclaim 31 wherein the compound is administered orally.
 33. The method ofclaim 31 wherein the compound is administered parenterally.
 34. Themethod of claim 31 wherein the compound is administered transdermally.35. The method of claim 31 wherein the compound is administered rectally36. The method of claim 31 wherein the compound is administered nasally.37. The method of claim 31 wherein the compound is administeredsublingually.
 38. The method of claim 31 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.39. The method of claim 31 wherein the compound is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


40. A method of treating an inflammatory disease selected from the groupconsisting of rheumatoid arthritis, asthma, and inflammatory boweldiseases, comprising administering to a subject with said disease aneffective amount of a compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 41. The method ofclaim 40 wherein the compound is administered orally.
 42. The method ofclaim 40 wherein the compound is administered parenterally.
 43. Themethod of claim 40 wherein the compound is administered transdermally.44. The method of claim 40 wherein the compound is administeredrectally.
 45. The method of claim 40 wherein the compound isadministered nasally.
 46. The method of claim 40 wherein the compound isadministered sublingually.
 47. The method of claim 40 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 48. The method of claim 40 wherein the compound is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


49. A method of treating a skin condition selected from the groupconsisting of wrinkles, lack of adequate skin firmness, lack of adequatedermal hydration and insufficient sebum secretion which comprisesadministering to a subject with said skin condition an effective amountof a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 50. The method ofclaim 49 wherein the compound is administered orally.
 51. The method ofclaim 49 wherein the compound is administered parenterally.
 52. Themethod of claim 49 wherein the compound is administered transdermally.53. The method of claim 49 wherein the compound is administeredtopically.
 54. The method of claim 49 wherein the compound isadministered rectally.
 55. The method of claim 49 wherein the compoundis administered nasally.
 56. The method of claim 49 wherein the compoundis administered sublingually.
 57. The method of claim 49 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 58. The method of claim 49 wherein the compound is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


59. A method of treating renal osteodystrophy comprising administeringto a subject with renal osteodystrophy an effective amount of a compoundhaving the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 60. The method ofclaim 59 wherein the compound is administered orally.
 61. The method ofclaim 59 wherein the compound is administered parenterally.
 62. Themethod of claim 59 wherein the compound is administered transdermally.63. The method of claim 59 wherein the compound is administeredrectally.
 64. The method of claim 59 wherein the compound isadministered nasally.
 65. The method of claim 59 wherein the compound isadministered sublingually.
 66. The method of claim 59 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 67. The method of claim 59 wherein the compound is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


68. A method of treating or preventing obesity of an animal, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in an animal comprising administering to an animal inneed thereof an effective amount of a compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 69. The method ofclaim 68 wherein the compound is administered orally.
 70. The method ofclaim 68 wherein the compound is administered parenterally.
 71. Themethod of claim 68 wherein the compound is administered transdermally.72. The method of claim 68 wherein the compound is administeredrectally.
 73. The method of claim 68 wherein the compound isadministered nasally.
 74. The method of claim 68 wherein the compound isadministered sublingually.
 75. The method of claim 68 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 76. The method of claim 68 wherein the compound is(20R)-2α-methyl-19,26,27-trinor-1α,25-dihydroxyvitamin D₃ having theformula:


77. The method of claim 68 wherein the animal is a human.
 78. The methodof claim 68 wherein the animal is a domestic animal.
 79. The method ofclaim 68 wherein the animal is an agricultural animal.